Environmental & Socio-Economic Studies

Environmental & Socio-economic Studies

DOI: 10.1515/environ-2015-0046

Micromorphological study (ultrastructure of lamina surface, seeds, ultrasculpture of pollen grains) of Gladiolus L. species (Iridaceae Juss.) of Ukrainian flora

Svitlana L. Zhygalova1, Oksana A. Futorna1,2, Anatoliy Levanets3

1Department of Systematics and Floristics of Vascular Plants, M.G. Kholodny Institute of Botany, National Academy of Sciences of Ukraine, Tereschenkivska Str. 2, Kyiv 01601, Ukraine 2O.V. Fomin Botanical Garden, Educational-Scientific Centre “Institute of Biology”, National Taras Schevchenko University of Kyiv, S. Petlyury Str. 1, Kyiv 01032, Ukraine 3Research Unit for Environmental Sciences and Management, Botany Department, North-West University, Potchefstroom, Republic of South Africa, Research Unit for Environmental Sciences and Management, P.O. Box X6001, North-West University 2520, Republic of South Africa E–mail address: (corresponding author) [email protected] ______ABSTRACT Micro-morphological characteristics of the four Gladiolus L. species of the Ukrainian flora (G. imbricatus L., G. italicus Mill., G. palustris Gaudin and G. tenuis M. Bieb.) as regards leaves, seeds and pollens are presented with this investigation in a detailed way. An examination of the surface structure of the leaves, seeds and pollen grains of the Gladiolus species indicates that the characteristics of the ultrastructure of leaves and of pollen grains are not diagnostic for distinguishing species, but they could be important at genus level (leaves: features such as being amphistomatic, having the same quantity of immersed stomata on both surfaces and having a high stomata index, the presence and localisation of papillae, the shape of epidermal cells; pollen grains: monosulcate type with two operculums). However, the type of surface ultrastructure of the seed coat is a diagnostic feature as at genus level so for species. It can be mentioned that propose the use of features such as the shape and position of the cicatricle, the type of cuticle, the shape and boundaries of cells of testa, and the anticlinal cell walls as diagnostic features at genera level. The shape of seeds, the presence and disposition of wing, the level of the periclinal cell walls of the seed coat and types of relief are additional diagnostic features for distinguishing of Gladiolus species.

KEY WORDS: Gladiolus, micromorphology, leaf lamina, seed, pollen ______

1. Introduction Micromorphological studies of the Iridaceae species lamina have been conducted by many The genus Gladiolus L. is the second largest authors (COLLINS, 1937; KARASAWA, 1942; SHORINA, genus of the family Iridaceae Juss after Iris L. 1975; RUDALL & MATHEW, 1990; RUDALL, 1991; According to the classification of GOLDBLATT & GOLDBLATT & LE THOMAS, 1997; EROL & KÜÇÜKER, MANNING (2008), the Gladiolus belongs to the 2003, 2007; KANDEMIR, 2009). However, the subfamily Crocoideae. The genus Gladiolus L. includes research data relating to the ultrastructure of the about 262 species that are found mainly in Africa leaves of the Ukrainian Gladiolus species are not and Madagascar, as well as in Europe, the Caucasus, available. central Asia, the Middle Asia and Asia Minor. Four The fruit and seed structure of Iridaceae was species are indicated for Ukrainian flora (MOSYAKIN studied by ARTYUSHENKO (1990) and RODIONENKO & FEDORONCHUK, 1999) – G. imbricatus L., G. italicus (1961). Later the seed micromorphology of 71 Mill., G. palustris Gaudin and G. tenuis M. Bieb. Iris species was investigated by Russian scientists Of these, Gladiolus imbricatus and G. tenuis are (RODIONENKO, 2005; ALEKSEEVA ET AL., 2007, 2011; noted as vulnerable in the RED DATA BOOK OF KRAVTSOVA & ZHYNKINA, 2008; ALEKSEEVA, 2010). UKRAINE (2009), and G. italicus and G. palustris are According to ALEKSEEVA ET AL. (2011), such considered endangered. researches are necessary because of the diagnostic

21 value of seed features at Iris species level. the Ukrainian flora and to investigate whether these MANNING & GOLDBLATT, (1991) studied the systematic characters are useful systematically. This is the first and phylogenetic significance of the seed testa of time such a study has been carried out in Ukraine. the African genera of Iridaceae – Nivenia Vent., Klattia Baker and Witsenia Thunb. EROL ET AL. 2. Materials and methods (2006) studied the seeds of nine Turkish Gladiolus species, including G. italicus. In Ukraine SZIKURA & The samples used for the study were SZIKURA (2003) detailed the characteristics of the herbarium specimens from the MG Kholodny fruit and seed testa of Iridaceae, including three Institute of Botany of NAS of Ukraine (KW). They Ukrainian Gladiolus species, but these results are were from a variety of habitats and were collected hardly reliable because of an incorrect selection in the wild. In order to study the ultrastructure of of samples (incorrect nomenclature and species the leaf lamina (middle third of leaf) and seeds, definition). the samples were mounted and sputter coated The literary data on the pollen morphology of with a thin layer of gold. To remove the wax from Iridaceae taxa is rather limited. RODIONENKO (1961) the leaf surface, each sample was placed in a xylol researched the pollen grains morphology of 73 solution for 24 hours. The study was carried out species of Iris, 24 species of Juno Tratt., five species using scanning electronic microscope JSM-6060 LA. of Crocus, one species of the genera Gynandriris The terminology used is, as far as possible, in Parl., Xiphium Mill. em. Rodion., Tigridia, accordance with works of ZAKHAREVICH (1954), Belamcanda, Sisyrinchium and five species of JUNIPER (1959), HALLAM (1970), HALLAM & CHAMBERS Gladiolus using light microscopy. The pollen grains (1970), CHUANG & HECKARD (1972), BARTHLOTT of Gladiolus, along with the size of the grains and (1981), JUNIPER & JEFFRY (1986), CHAKRABARTY & of the exine cells, are detailed by the author in a MUKHERJEE (1986), STERN (1992), BARTHLOTT ET AL. table. GOLDBLATT & LE THOMAS (1997) discussed the (1998). For the SEM study of the pollen grains’ phylogenetic reconstruction and classification of ultrasculpture (CEM, JSM 6060 LA), the samples the Afro-Madagascan genus Aristea after examining were mounted in 96% ethanol and then sputter the pollen grains using light and scanning electron coated with gold. The pollen grains were also microscopy. The ultrasculpture of pollen grains of examined under the light microscope (LM), 29 taxa of the genus Crocus were also studied by without acetolysis, and mounted in 30% glycerin Turkish scientists (IŞIK & DÖNMEZ, 2006; CANDAN (stained by fuchsine), but after rehydration of the ET AL., 2009; CANDAN & ÖZHATAY, 2013; and others). anthers in 50% glycerin. The pollen grains were These authors showed that the ultrasculpture of described using the terminology of PUNT ET AL. the pollen grains of the Turkish Crocus species is (1994) and TOKAREV (2002). similar to Europe’s Crocus species. The pollen morphology of 10 Iris species from the Far East 2.1. List of samples was examined by BOLTENKOV & GRIGORJEVA (2012) using a light and scanning electron microscope. G. іmbricatus L.: 1. Chernivtsi Region, Ghertsaiv However, the data on the pollen morphology District, between villages Kulykivka and Tarnavka, of the Gladiolus species is limited. BEUG (1963) meadow. 03.07.2005. A. Tokaryuk, O. Volutsa (KW), described the pollen grains of five Gladiolus 2. Ivano-Frankivsk Region, Nadvirnyansk District, species and, as mentioned above, RODIONENKO east border of village Mykulychin, meadow, 1059 m also studied the pollen grains of five species. a.s.l. 24.08.2010. L. Borsukevich, K. Danylyuk (KW), Furthermore, BOBROV ET AL. (1983) characterised 3. Chernihiv Region, Oster District, environs of the pollen grains of G. imbricatus, while the village Yevminka, meadow along the Desna River. palynomorphological data of Gladiolus is cited in 24.06.2007. O. Bulakh, S. Zhygalova (KW), 4. Sumy the monograph “The Iris family” (GOLDBLATT & Region, Putivl District, beam near village Shyryajevo. MANNING, 2008). However, such research is 21.06.2009. Ya. Didukh (KW), 5. Transcarpathian absent in Ukraine. We have already studied the Region, urban village Jasinya, wet meadow at the micromorphological characteristics of G. imbricatus hill. 19.07.2008. I. Olshanskij, S. Zhygalova (KW). of Ukrainian flora (ZHYGALOVA & FUTORNA, 2013) and our investigation indicated the necessity of G. tenuis M. Bieb.: 1. Poltava Region, Kobelyaky complex micromorphological study of all Ukrainian District, environs of the biostation “Luchki” of the Gladiolus species. Poltava Pedagogical Institute, reserve tract The aim of this study is to give a complete “Sokilske”, meadow after logged land. 18.06.1994. description of the surface structure of leaves, N.O. Stetsyuk (KW), 2. Chernigiv Region, Sosnytsya seeds and pollen grains of the Gladiolus species of District, 1 km on the west from the village 22

Yaklychi, meadow (with dominating Festuca G. palustris Gaudin.: Ivano-Frankivsk Region, pratensis and Deschampsia cespitosa) on the flood Halych District, village Bovshiv, m. Kassova. May land of Desna River. 28.06.2006. O.V. Lukash (KW), 1979. Ya. Didukh (KW). 3. Sumy Region, Burin District, water meadow of the Sejm River, 2 km from village Pisky (down- 3. Results stream). 05.07.2005. M.S. Kozyr (KW), 4. Sumy Region, near village Seredyna-Buda, wet meadow, The ultrastructural features of leaf lamina surfaces. riverhead of the Bobryk River, to the north-west The study of the ultrastructure of the leaves of the village Sorokine. 25.07.2011. O.F. Podejko, determined that the lamina of all Gladiolus species S.M. Panchenko (KW), 5. Kharkiv Region, Pechenigy is similar. The leaves are therefore amphistomatic. District, the source of beam Hnylushka, swampy Immersed stomata (the brahyparacytic type) meadow. 24.06.1996. I.A. Korotchenko (KW). orientate by a longer axle along the lamina. The quantity of stomata on both surfaces of the G. italicus Mill.: 1. Crimea, Yalta reserve, Shtanhejevska lamina is nearly the same (difference within the pathway, stone hills, gravel soil. 02.06.2001. Ya. error). The stomata index was high in all the Didukh (KW), 2. Crimea, hollow around the samples studied; it was not dependent on plant observatory. 15.06.1918. S. Stankov (KW), 3. habitats. It is noted papillae on the both sides of Crimea, Sevastopol g/s, village Reservne, near the lamina (Fig. 1B), which tend to have dense lines field. 17.06.1980. Ya. Didukh (KW). along the proximal walls of the epidermal cells.

A B Fig. 1. Types of relief of abaxial surface of G. imbricatus: A: colliculate; B: colliculate-mesh

The leaf surface is characterised by winding the both sides. We find out the presence of two type (cells between nerves) or straight (cells upon the of wax: wax plates (between the nerves) and wax nerves) and elongated projections of epidermal crusts (upon the nerves and at border of lamina) cells on both sides. All studied samples were (Fig. 2A, B). The leaves of G. palustris are mostly characterised by colliculate-mesh (between the characterized by the presence of wax crusts (Fig. 2C). nerves) and colliculate (upon the nerves and at The examination of the leaves’ ultrastructure the border of the lamina) relief of epidermal tissue determined that the leaf lamina of all Gladiolus (Figures 1A, B). The anticlinal walls of epidermal species can be characterised as: amphistomatic; cells are evenly thickened; the periclinal walls of immersed stomata brahyparacytic type; high epidermal cells are concave. stomata index; rugose type of cuticle; winding or The Gladiolus species we studied, which were straight shape and elongated projections of from different habitats in Ukraine, were epidermal cells; colliculate-mesh and colliculate characterised by the rugose type of cuticle; the relief; evenly thickened anticlinal walls of epidermal cuticle ramparts around the stomata are easily seen. cells; concave walls of periclinal epidermal cells; The wax well develops on the all leaf surface on and the presence of wax plates and wax crusts. 23

A B Fig. 2. Types of wax: A: wax plates (G. imbricatus); B: wax crusts (G. imbricatus); C: wax crusts (G. palustris)

The seeds’ ultrastructural features. Both common or prolonged obovate (G. palustris), pyriform or and distinctive features characterise the seeds of globosepyriform (G. italicus). Gladiolus imbricatus, plants of the Gladiolus species. The common features G. tenuis and G. palustris have seeds with wing. are: the shape and position of the cicatricle (square, Seeds of G. italicus are without wing. The periclinal small, by position – basal); the type of cuticle cell walls of the seed coat are different: convex (rugose, well developed); the cells of testa are (on wing and sometimes on the seed body of polygonal and their boundaries are clearly seen; G. imbricatus and G. tenuis), flat or concave (on and the anticlinal cell walls are always uniformly the seed body of G. imbricatus and G. tenuis), or thickened and straight. These characters of seed concave (G. italicus and G. palustris). Different testa could be diagnostic at genera level. types of relief characterise the ultrastructure of The distinct characteristics are: the shape of Gladiolus seeds: colliculate (on wing of G. imbricatus the seeds, the presence of wing; the level of the and G. tenuis) (Fig. 3A), rugose (on the seed body periclinal cell walls of the seed coat; and the types of G. imbricatus and G. tenuis) (Fig. 3B), reticulate- of relief. In shape, the seeds are obconic or prolonged cellulate (G. italicus and G. palustris, sometimes on obovate (G. imbricatus), obovoideum (G. tenuis) the seed body of G. imbricatus and G. tenuis) (Fig. 3C).

A B C Fig. 3. The relief types of the seed testa: A: colliculate (G. tenuis (on wing)); B: rugose (G. tenuis (on seed body)); C: reticulate-cellulate (G. palustris)

The pollen grains’ ultrasculptural features. It is indeed characterised by the monosulcate type, found that the common features that characterise with the presence of two operculums (Fig. 4A). the pollen grains of Ukrainian Gladiolus species, Furrow with more or less distinct smooth edges, as indicated by all investigated Gladiolus species, covered with ornamental membranes. The sculpture are: the pollen grains are monads, large (51-100 μm) of pollen grains on the proximal side and between and heteropolar. The shape varies from oblate to the furrows is spinulose (all the species) (Fig. 5C), oblate-spheroid. The outline of pollen grains on sometimes perforated (G. italicus, G. tenuis) (Fig. 5A), the equatorial side is elliptic (Fig. 4B). The pollen with rare tubercles (G. italicus, G. tenuis, G. palustris) grains of all investigated Gladiolus species were (Fig. 5B).

A B Fig. 4. The general view of G. imbricatus pollen grain: A: SEM; B: LM, x 40

A B C Fig. 5. The ultrasculpture on the proximal side of pollen grains: A: spinulose, perforate, with rare tubercles sculpture (G. tenuis); B: tubercle on spinulose sculpture (G. italicus); C: spinulose sculpture (G. imbricatus)

4. Discussion endemic Crocus from the flora of Turkey showed the absence of wax and papillae on the leaf surface Therefore, having conducted a detailed (EROL & KÜÇÜKER, 2007). We suppose the presence micromorphological study, it can be concluded and localisation of papillae on the Gladiolus species that common features characterise the leaf leaf surface could be important at genus level. surface of the Ukrainian Gladiolus species. Features EROL & KÜÇÜKER, (2007) found that the epidermal such as being amphistomatic, having the same cells of Crocus leaves were characterised by a quantity of immersed stomata on both surfaces and straight shape. Our study showed winding or having a high stomata index were characteristic straight and elongated projections of epidermal of plants growing in places with a high level of cells on both sides. The shape of epidermal cells insolation (EZAU, 1980; NAGALEVSKIJ & NIKOLAYEVSKIJ, is a diagnostic feature for many Monocotyledons 1981; VASILJEV, 1988; EVERT, 2006). In our opinion, and Dicotyledons (EZAU, 1980; EVERT, 2006). Thus sword-shaped leaves that get much sun radiation this feature could also be an additional diagnostic on both sides explains the presence of xeromorphic feature for distinguishing Iridaceae genera. The features. We suggest these peculiarities could be relief type of epidermal tissue of leaf lamina is an diagnostic at the genus level. exceedingly stable characteristic (JUNIPER & JEFFRY, Much study has been done on the structure, 1986; JAYEOLA & THORPE, 2000). Our research the type and localisation of trichomes, which are confirms this. used as stable diagnostic features for many taxa The degree of wax layer development depends (EZAU, 1980; EVERT, 2006). However, virtually no on the species’ origin and on ecological growth data is available concerning the structure of the conditions. In the opinion of some researchers, triсhomes of Iridaceae in particular. Many the wax and cuticle develop better in extreme researchers have paid attention to the structure environmental conditions (EZAU, 1980; NAGALEVSKYJ of the glandular hairs on the flower tepals of & NIKOLAYEVSLIJ, 1981; EVERT, 2006). Other scientists some Iridaceae taxa. The leaf anatomy of some think that the type and the quantity of wax are

25 diagnostic features (DALY, 1964). Our study showed characterised by the monosulcate type, but with that the wax well develops on the all leaf surface the presence of two operculums (Figure 4A). on the both sides in all samples of Ukrainian Our data therefore confirms the study of Gladiolus species. Thus, we can conclude that GOLDBLATT & MANNING (2008), which indicates a characteristics of the leaves’ ultrastructure are basic monosulcate type with a two-banded not diagnostic for distinguishing species, but they operculum, ancestral for Crocoideae. It is therefore could be important at the genus level. suggested that the peculiarities of the ultrasculpture The seeds testa features are conservative and of the pollen grains of the Gladiolus species of stable, and seem to depend minimally on Ukrainian flora could be a diagnostic feature at ecological factors (BARTHLOTT, 1981; KHONCHAROVA, the genera level. 2006). A study of the seeds coat ultrastructure of the Gladiolus species showed both common and 5. Conclusions distinct features that characterise the seeds. It is proposed the use of features such as the shape Therefore, having conducted a detailed and position of the cicatricle, the type of cuticle, micromorphological study, it can be concluded the shape and boundaries of cells of testa, and the that common features characterise the leaf surface anticlinal cell walls as diagnostic features at and pollen grains of the Ukrainian Gladiolus species. genera level. It is suggested these peculiarities could be A comparison of our results with published diagnostic at the genus level. A study of the seeds data on the structure of the seeds of other species coat ultrastructure of the Gladiolus species showed of the genus Gladiolus (EROL ET AL., 2006) indicate both common and distinct features that characterise that the type of surface ultrastructure of seed coat the seeds. It is proposed the use of features such might serve as a diagnostic feature for species. Our as the shape and position of the cicatricle, the study of Ukrainian Gladiolus species showed that type of cuticle, the shape and boundaries of cells in particular the shape of seeds, the presence and of testa, and the anticlinal cell walls as diagnostic disposition of wing, the level of the periclinal cell features at genera level. The shape of seeds, the walls of the seed coat and types of relief are presence and disposition of wing, the level of the additional diagnostic features for distinguishing periclinal cell walls of the seed coat and types of of Gladiolus species. relief are additional diagnostic features for The peculiarities of pollen grains is one of distinguishing of Gladiolus species. most stable diagnostic criteria for identification or differentiation of families, genera, sections and References species (especially morphologically close) (ERDTMAN, 1952; FERGUSON, 1985; NAKAMURA, 1943). However, Alekseeva N.B. 2010. Seed coat morphology of some species as mentioned above, the published results of of Iris (Iridaceae) in the context of the genus taxonomy. Bot. zhurnal, 3 (95): 345-350. previous palynomorphological investigations yielded Alekseeva N.B., Boltenkov Ye.V.,Mironova L.N. 2011. Some little information concerning the peculiarities of peculiarities of seed morphology of the Iris (Iridaceae) the pollen grains of the Gladiolus species. BOBROV species from the Russian Far East. Bot. zhurnal, 7 (96): ET AL. (1983) characterised the pollen grains of G. 851-857. Alekseeva N.B., Mironova L.N. 2007. Critical notes about imbricatus. The description of the pollen grains of some species of genus Iris (Iridaceae) in Siberia and Mediterranean species G. illyricus is presented on Russian Far East. Bot. zhurnal, 6 (92): 916-925. the palynomorphological database PALDAT (2000). Artyushenko Z.T. 1990. The atlas on the describing morphology of The palynological investigations of the Gladiolus the vascular plants. Nauka Publ. house, Leningrad. species of the Ukrainian flora were carried out in Barthlott W. 1981. Epidermal and seed surface characters of plants: systematic applicability and some evolutionary Ukraine for the first time in this study. aspects. Nordic J. of Bot., 1 (3): 345-355. Some of our results are not consistent with Barthlott W., Neinhuis C., Cutler D., Ditsch F., Meusel I., previous results of other authors. According to Theisen I., Wilhelmi H. 1998. Classification and terminology clasic palynological works (ERDTMAN, 1952; SLADKOV, of plant epicuticular waxes. Bot. J. of the Linnean Soc., 126 (3): 237-260. 1967; BOBROV ET AL., 1983), the sulcate type of Beug H.-J. 1963. Leitfaden der Pollen bestimmung fur pollen grains is the most prevalent type of Mitteleuropa und angrenzende Gebiete. Lieferung 1. Gustav Monocotyledons pollen grains. Concerning the Fischer Verlag, Jena. Gladiolus species, the authors noted a monosulcate Bobrov A.Ye., Kupriyanova L.A., Lytvinceva M.V., Tarasevich type with one operculum (mostly after studying V.F. 1983. The spores of Polypodiophyta and the pollen of Pinophyta and Monocotyledons of the flora of European the pollen grains of G. imbricatus). Against those part of the USSR. Nauka Publ. house, Leningrad. results, our study showed that the pollen grains of all investigated Gladiolus species were indeed 26

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